Limits...
Enhanced intracellular dissociation of major histocompatibility complex class I-associated peptides: a mechanism for optimizing the spectrum of cell surface-presented cytotoxic T lymphocyte epitopes.

Sijts AJ, Pamer EG - J. Exp. Med. (1997)

Bottom Line: We find that p60 449-457-H2-K(d) complexes retained intracellularly with brefeldin A have a half-life of 30 min, and thus are less stable than surface complexes.We find that intracellular H2-K(d) molecules can bind new CTL epitopes for up to 3 h after their synthesis.Our studies provide a glimpse of peptide interaction with MHC class I molecules in the endoplasmic reticulum/proximal Golgi complex of intact, infected cells.

View Article: PubMed Central - PubMed

Affiliation: Section of Infectious Diseases and Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
Association of antigenic peptides with newly synthesized major histocompatibility complex (MHC) class I molecules occurs in the endoplasmic reticulum and is a critical early step for the initiation of cytotoxic T lymphocyte (CTL)-mediated immune defenses. Pathogen-derived peptides compete with a plethora of endogenous peptides for MHC class I grooves. We find that two H2-K(d)-restricted peptides, which derive from the Listeria monocytogenes p60 antigen, accumulate in infected cells with different kinetics. Although competition assays suggest that both epitopes are bound with equivalent affinity, they dissociate from MHC class I molecules at markedly different rates. p60 217-225 forms complexes with H2-K(d) with a half-life >6 h, while p60 449-457 dissociates from H2-K(d) with a half-life of approximately 1 h. We find that p60 449-457-H2-K(d) complexes retained intracellularly with brefeldin A have a half-life of 30 min, and thus are less stable than surface complexes. While peptide dissociation from retained MHC class I molecules is enhanced, retained H2-K(d) molecules maintain a remarkable capacity to bind new T cell epitopes. We find that intracellular H2-K(d) molecules can bind new CTL epitopes for up to 3 h after their synthesis. Our studies provide a glimpse of peptide interaction with MHC class I molecules in the endoplasmic reticulum/proximal Golgi complex of intact, infected cells. We propose that the increased intracellular lability of peptide-MHC class I complexes may function to optimize the spectrum of peptides presented to T lymphocytes during cellular infection.

Show MeSH

Related in: MedlinePlus

H2-Kd trafficking in CHX–ANM-treated J774 cells. J774  cells were pulse labeled in the presence of 5 μg/ml BFA to retain class I  molecules, and then chased in the presence of CHX (50 μg/ml) and  ANM (30 μg/ml). BFA was removed from the culture medium immediately after the pulse, and cells were either harvested immediately (0 h) or  chased 1, 2, or 3 h in medium with or without CHX and ANM. H2-Kd  molecules were immunoprecipitated, mock- or endo H–digested, and analyzed by SDS PAGE. Molecular weight markers are indicated on the left.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196277&req=5

Figure 5: H2-Kd trafficking in CHX–ANM-treated J774 cells. J774 cells were pulse labeled in the presence of 5 μg/ml BFA to retain class I molecules, and then chased in the presence of CHX (50 μg/ml) and ANM (30 μg/ml). BFA was removed from the culture medium immediately after the pulse, and cells were either harvested immediately (0 h) or chased 1, 2, or 3 h in medium with or without CHX and ANM. H2-Kd molecules were immunoprecipitated, mock- or endo H–digested, and analyzed by SDS PAGE. Molecular weight markers are indicated on the left.

Mentions: Most H2-Kd molecules exit the ER within 1 to 2 h after synthesis, indicating that they have bound a peptide during this time interval. Therefore, our finding that retained H2Kd molecules bind newly generated p60 217-225 and p60 449-457 for up to 3 h suggests that some of the H2-Kd molecules binding the L. monocytogenes epitopes previously bound other peptides. A potential problem with this interpretation is that protein synthesis inhibition, while preventing new H2-Kd synthesis, may also inhibit production of endogenous peptides from newly synthesized proteins. This would be particularly true if defective ribosomal products (6) are a major source of MHC class I–associated peptides. Thus, inhibiting protein synthesis may lead to a paucity of endogenous peptides and an abundance of empty H2-Kd molecules in the ER, thereby enhancing bacterial peptide capture. The importance of endogenous peptide production for normal H2-Kd trafficking to the cell surface can be readily demonstrated with proteasome inhibitors, which inhibit cytosolic peptide generation and retard H2-Kd trafficking to the cell surface (18). To address the issue of peptide production when host cell protein synthesis is inhibited, we pulse labeled BFA-treated J774 cells with [35S]methionine and chased for 1, 2, and 3 h in the presence of CHX– ANM. Despite inhibition of cellular protein synthesis, pulse-labeled H2-Kd molecules acquired endo H resistance upon BFA removal, indicating that they trafficked through the Golgi (Fig. 5). Thus, sufficient quantities of peptide are generated in the absence of host cell protein synthesis to enable H2-Kd transit with essentially normal kinetics. Importantly, all detectable H2-Kd molecules trafficked through the Golgi within 2 h after release of the BFA blockade, indicating that within this time period they had acquired an endogenous peptide. In contrast, H2-Kd molecules retained intracellularly with BFA continued to acquire L. monocytogenes epitopes 2–3 h after H2-Kd synthesis (Fig. 4). These findings suggest that H2-Kd molecules, if retained in a cellular compartment in which peptide loading occurs, are capable of binding peptides for a period of time that exceeds the time necessary for the acquisition of endogenous peptides.


Enhanced intracellular dissociation of major histocompatibility complex class I-associated peptides: a mechanism for optimizing the spectrum of cell surface-presented cytotoxic T lymphocyte epitopes.

Sijts AJ, Pamer EG - J. Exp. Med. (1997)

H2-Kd trafficking in CHX–ANM-treated J774 cells. J774  cells were pulse labeled in the presence of 5 μg/ml BFA to retain class I  molecules, and then chased in the presence of CHX (50 μg/ml) and  ANM (30 μg/ml). BFA was removed from the culture medium immediately after the pulse, and cells were either harvested immediately (0 h) or  chased 1, 2, or 3 h in medium with or without CHX and ANM. H2-Kd  molecules were immunoprecipitated, mock- or endo H–digested, and analyzed by SDS PAGE. Molecular weight markers are indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196277&req=5

Figure 5: H2-Kd trafficking in CHX–ANM-treated J774 cells. J774 cells were pulse labeled in the presence of 5 μg/ml BFA to retain class I molecules, and then chased in the presence of CHX (50 μg/ml) and ANM (30 μg/ml). BFA was removed from the culture medium immediately after the pulse, and cells were either harvested immediately (0 h) or chased 1, 2, or 3 h in medium with or without CHX and ANM. H2-Kd molecules were immunoprecipitated, mock- or endo H–digested, and analyzed by SDS PAGE. Molecular weight markers are indicated on the left.
Mentions: Most H2-Kd molecules exit the ER within 1 to 2 h after synthesis, indicating that they have bound a peptide during this time interval. Therefore, our finding that retained H2Kd molecules bind newly generated p60 217-225 and p60 449-457 for up to 3 h suggests that some of the H2-Kd molecules binding the L. monocytogenes epitopes previously bound other peptides. A potential problem with this interpretation is that protein synthesis inhibition, while preventing new H2-Kd synthesis, may also inhibit production of endogenous peptides from newly synthesized proteins. This would be particularly true if defective ribosomal products (6) are a major source of MHC class I–associated peptides. Thus, inhibiting protein synthesis may lead to a paucity of endogenous peptides and an abundance of empty H2-Kd molecules in the ER, thereby enhancing bacterial peptide capture. The importance of endogenous peptide production for normal H2-Kd trafficking to the cell surface can be readily demonstrated with proteasome inhibitors, which inhibit cytosolic peptide generation and retard H2-Kd trafficking to the cell surface (18). To address the issue of peptide production when host cell protein synthesis is inhibited, we pulse labeled BFA-treated J774 cells with [35S]methionine and chased for 1, 2, and 3 h in the presence of CHX– ANM. Despite inhibition of cellular protein synthesis, pulse-labeled H2-Kd molecules acquired endo H resistance upon BFA removal, indicating that they trafficked through the Golgi (Fig. 5). Thus, sufficient quantities of peptide are generated in the absence of host cell protein synthesis to enable H2-Kd transit with essentially normal kinetics. Importantly, all detectable H2-Kd molecules trafficked through the Golgi within 2 h after release of the BFA blockade, indicating that within this time period they had acquired an endogenous peptide. In contrast, H2-Kd molecules retained intracellularly with BFA continued to acquire L. monocytogenes epitopes 2–3 h after H2-Kd synthesis (Fig. 4). These findings suggest that H2-Kd molecules, if retained in a cellular compartment in which peptide loading occurs, are capable of binding peptides for a period of time that exceeds the time necessary for the acquisition of endogenous peptides.

Bottom Line: We find that p60 449-457-H2-K(d) complexes retained intracellularly with brefeldin A have a half-life of 30 min, and thus are less stable than surface complexes.We find that intracellular H2-K(d) molecules can bind new CTL epitopes for up to 3 h after their synthesis.Our studies provide a glimpse of peptide interaction with MHC class I molecules in the endoplasmic reticulum/proximal Golgi complex of intact, infected cells.

View Article: PubMed Central - PubMed

Affiliation: Section of Infectious Diseases and Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
Association of antigenic peptides with newly synthesized major histocompatibility complex (MHC) class I molecules occurs in the endoplasmic reticulum and is a critical early step for the initiation of cytotoxic T lymphocyte (CTL)-mediated immune defenses. Pathogen-derived peptides compete with a plethora of endogenous peptides for MHC class I grooves. We find that two H2-K(d)-restricted peptides, which derive from the Listeria monocytogenes p60 antigen, accumulate in infected cells with different kinetics. Although competition assays suggest that both epitopes are bound with equivalent affinity, they dissociate from MHC class I molecules at markedly different rates. p60 217-225 forms complexes with H2-K(d) with a half-life >6 h, while p60 449-457 dissociates from H2-K(d) with a half-life of approximately 1 h. We find that p60 449-457-H2-K(d) complexes retained intracellularly with brefeldin A have a half-life of 30 min, and thus are less stable than surface complexes. While peptide dissociation from retained MHC class I molecules is enhanced, retained H2-K(d) molecules maintain a remarkable capacity to bind new T cell epitopes. We find that intracellular H2-K(d) molecules can bind new CTL epitopes for up to 3 h after their synthesis. Our studies provide a glimpse of peptide interaction with MHC class I molecules in the endoplasmic reticulum/proximal Golgi complex of intact, infected cells. We propose that the increased intracellular lability of peptide-MHC class I complexes may function to optimize the spectrum of peptides presented to T lymphocytes during cellular infection.

Show MeSH
Related in: MedlinePlus